5 January 2010. A synapse-promoting protein called LRRTM2 has been identified as a ligand for neurexin, a strong candidate susceptibility gene for schizophrenia, autism, and other neuropsychiatric disorders (see SRF related news story). This newly discovered LRRTM2-neurexin interaction, described in two reports published in Neuron on December 24, adds to the list of diverse partnerships between cell adhesion molecules that bridge across neurons to regulate how synapses are established.

The two studies—one from Thomas Südhof and colleagues at Stanford University and one from Anirvan Ghosh's group at the University of California, San Diego—come swiftly after the initial identification of LRRTM (that's leucine-rich repeat transmembrane) proteins as stimulators of synapse formation in cell culture by Anne Marie Craig's group at the University of British Columbia (Linhoff et al., 2009). LRRTMs comprise a family of four proteins found in neurons that span the cell membrane and protrude into the extracellular space with a long stretch of leucines, a structure predisposed to interacting with other proteins. A variant in the gene for the LRRTM1 type has been linked to schizophrenia (Francks et al., 2007 and Ludwig et al., 2009), and although the new findings concern LRRTM2 specifically, they reiterate the theme that problems with synapse assembly and maintenance can lead to psychiatric disorders (Südhof, 2008).

Using complementary and sometimes overlapping methods, the new studies place LRRTM2 on the receiving, postsynaptic end of a developing synapse. When LRRTM2 binds the extracellular part of neurexin located in a contacting axon, this triggers excitatory synapse formation, as documented by increases in synapse density, size, glutamate receptor aggregation, and synaptic currents. Although similar to the synapse-promoting bridge formed between neuroligins and neurexins, LRRTMs are unrelated to neuroligins, and as Südhof's group found, somewhat more discerning: LRRTM2 bound only those neurexin proteins that lacked a specific splice site, while neuroligins bind neurexins both with and without this site. This difference underscores a variety of possible interactions between cell adhesion molecules, which can contribute to specifying the diverse synapse types in the brain.

Artificial synapses
Similar to Craig's study, first author Jaewon Ko and colleagues in Südhof's lab initially probed the synapse-promoting properties of LRRTM2 by seeing if they could trick a neuron into forming an artificial synapse with a non-neuronal cell made to express LRRTM2. After two days, the LRRTM2-containing cells sported synapse-like junctions that stained positively for presynaptic markers of vesicles containing glutamate, but not the inhibitory neurotransmitter GABA. This indicated that LRRTM2 induced specifically excitatory synapses. To verify this with real synapses, they overexpressed LRRTM2 in cultured neurons and also found an increase in excitatory, but not inhibitory, synapse density and size.

Next, the researchers looked for LRRTM2's presumed partner in triggering synapse formation. Using affinity chromatography, the researchers trolled a mixture of brain proteins with LRRTM2 to trap those that could stick tightly to LRRTM2. They pulled out both α and β forms of neurexin, a known presynaptic protein. Neurexins bound LRRTM2 tightly, even when LRRTM2 was on the cell surface, indicating a direct interaction between the two proteins involving their extracellular ends. Further analysis revealed the sensitivity of LRRTM2 to splice site #4 on both forms of neurexin. The LRRTM2-neurexin interaction was critical to artificial synapse formation because adding free-floating neurexin—which interferes with the access of axon-tethered neurexins to LRRTM2—substantially reduced synapse formation.

A functional focus
The study by Ghosh, first author Joris de Wit, and colleagues also begins with an artificial synapse assay, but once the researchers found that LRRTM2 induced more synapse-like structures than the other members of the LRRTM family, they were quickly tinkering with endogenous LRRTM2 in hippocampal neurons. When the researchers introduced a silencing RNA molecule directed to LRRTM2 to lower endogenous levels of LRRTM2 in cultured neurons, they found a 40 percent decrease in synapse density in only excitatory synapses; inhibitory synapse formation was unchanged.

In addition to inducing presynaptic specializations, LRRTM2 also seemed to organize the post-synaptic side of things. Knocking down LRRTM2 levels in neurons led to a 33 percent decrease in the density of GluR1 protein—a subunit of the AMPA type of glutamate receptor—on the cell surface, and the extracellular portion of LRRTM2 colocalized to several other glutamate receptor subunits introduced into non-neuronal cells. Meanwhile, the intracellular portion of LRRTM2 colocalized to PSD-95, a synaptic scaffolding protein found exclusively in excitatory synapses, suggesting that LRRTM2 assembles post-synaptic elements both above and below the cell membrane. Then came an acid test: Could decreasing LRRTM2 tamper with excitatory synapse function in vivo? To address this, the researchers injected a lentivirus containing the LRRTM2-lowering RNA molecule into the hippocampus of young rats. A week or so later, hippocampal slices were cut and simultaneous recordings of infected and uninfected dentate gyrus neurons showed that the infected ones—with less LRRTM2—had a 58 percent reduction in synaptic currents flowing through AMPA receptors when compared to uninfected cells with normal amounts of LRRTM2; a 54 percent reduction was measured for NMDA currents. These results argue that loss of LRRTM2 decreases glutamatergic neurotransmission in vivo.

The group then turned their sights to identifying a partner for LRRTM2, whose extracellular end they determined to be crucial for forming synapses. They, too, found that neurexins readily bound LRRTM2, and further experiments winnowed down LRRTM2's binding partners to the neurexin 1α and 1β isoforms. The study also concluded by showing that neurexin was critical to the synapse-forming abilities of LRRTM2 in the artificial synapse assay, using a technique different from Südhof's study: when an RNA construct that lowered neurexin 1 expression was introduced into the neurons, the non-neuronal cells expressing LRRTM2 could no longer induce synapses.

The different approaches in these two studies converge onto a picture of a new partnership between cell adhesion molecules: when post-synaptic LRRTM2 binds to presynaptic neurexin, excitatory synapses emerge. The exact nature of this synapse-promoting ability remains unclear, however. Rather than instigating the initial contact between two neurons, the LRRTM2-neurexin interaction could be critical for organizing or stabilizing transient synapses that are initially formed by another independent mechanism. What is clear is that there are multiple cell adhesion pathways operating in parallel between neurons, and these may specify the wide range of synapse types in the brain, or even provide redundancy. The complexities of this synaptic apparatus also point to more ways to disrupt—as well as potentially fix—synapse formations that may go awry in brain disorders like schizophrenia and autism.—Michele Solis.

One Hundred Years of Insanity: The Relationship Between Schizophrenia and Autism
The great Colombian author Gabriel García Márquez reified the cyclical nature of history in his Nobel Prize-winning 1967 book, One Hundred Years of Solitude. Eugen Bleuler’s less-famous book Dementia Præcox or the Group of Schizophrenias, originally published in 1911, saw first use of the term “autism,” a form of solitude manifest as withdrawal from reality in schizophrenia. This neologism, about to celebrate its centenary, epitomizes an astonishing cycle of reification and change in nosology, a cycle only now coming into clear view as molecular-genetic data confront the traditional, age-old categories of psychiatric classification.

The term autism was, of course, redefined by Leo Kanner (1943) for a childhood psychiatric condition first considered as a subset of schizophrenia, then regarded as quite distinct (Rutter, 1972) or even opposite to it (Rimland, 1964; Crespi and Badcock, 2008), and most recently seen by some researchers as returning to its original Bluelerian incarnation (e.g., Carroll and Owen, 2009). An outstanding new paper by McCarthy et al. (2009), demonstrating that duplications of the CNV locus 16p11.2 are strongly associated with increased risk of schizophrenia, has brought this question to the forefront of psychiatric inquiry, because deletions of this same CNV are one of the most striking recently-characterized risk factors for autism. Additional CNVs, such as those at 1q21.1 and 22q11.21 have also been associated with autism and schizophrenia in one or more studies (e.g., Mefford et al., 2008; Crespi et al., 2009; Glessner et al., 2009), which has led some authors to infer that since an overlapping set of loci mediates risk of both conditions, autism and schizophrenia must be more similar than previously conceived (e.g., Carroll and Owen, 2009; Guilmatre et al., 2009). Similar considerations apply to several genes, such as CNTNAP2 and NRXN1, various disruptions of which have likewise been linked with both conditions (Iossifov et al., 2008; Kirov et al., 2008; Burbach and van der Zwaag, 2009).

So does this plethora of new molecular-genetic data imply that Blueler was indeed correct, if not prescient, that autism and schizophrenia are manifestations of similar disease processes? The answer may appear tantalizingly close, but will likely remain inaccessible without explicit consideration of alternative hypotheses and targeted tests of their differentiating predictions. This approach is simply Platt’s (1964) classic method of strong inference, which has propelled molecular biology so far and fast but left psychiatry largely by the wayside (Cannon, 2009). The alternative hypotheses in this case are clear: with regard to causation from specific genetic and genomic risk factors, autism and schizophrenia are either: 1) independent and discrete, 2) partially yet broadly overlapping, 3) subsumed with autism as a subset of schizophrenia, or 4) diametrically opposite, with normality in the centre. CNVs are especially useful for testing among such alternative hypotheses, because they naturally involve highly-penetrant perturbations in two opposite directions, due to deletions vs duplications of more or less the same genomic regions. Hypotheses 2), 3) and 4) thus predict that autism and schizophrenia should share CNV risk loci, but 2) and 3) predict specific rearrangements (deletions, duplications, or both) shared across both conditions; by contrast, hypothesis (4) predicts that, as highlighted by McCarthy et al. (2009), reciprocal CNVs at the same locus should mediate risk of autism versus schizophrenia. This general approach was pioneered by Craddock et al. (2005, 2009), in their discussion of explicit alternative hypotheses for the relationship between schizophrenia and bipolar disorder, which are now known to share a notable suite of risk alleles.

A key assumption that underlies tests of hypotheses for the relationship between autism and schizophrenia is accuracy of diagnoses. For schizophrenia, this is seldom at issue. However, diagnoses of autism, or autism spectrum disorders such as PDD-NOS, are normally made at an age well before the first manifestations of schizophrenia in adolescence or early adulthood, which generates a risk for false-positive diagnoses of premorbidity to schizophrenia as autism or autism spectrum (e.g., Eliez, 2007). The tendencies for males to exhibit worse premorbidity to schizophrenia than females (Sobin et al., 2001; Tandon et al., 2009), for CNVs to exert severe effects on diverse aspects of early neurodevelopment (Shinawi et al., 2009), and for schizophrenia of earlier onset to exhibit a higher male sex-ratio bias and a stronger tendency to be associated with CNVs rather than other causes (Remschmidt et al., 1994; Rapoport et al., 2009), all suggest a high risk for false-positive diagnoses of autistic spectrum conditions in individuals with these genomic risk factors (Feinstein and Singh, 2007; Reaven et al., 2008; Sugihara et al., 2008; Starling and Dossetor, 2009). Possible evidence of such risk comes from diagnoses of autism spectrum conditions in children with deletions at 15q11.2, 15q13.3, and 22q11.21, and duplications of 16p11.2, CNVs for which schizophrenia risk has been well established from studies of adults (Antshel et al., 2007; Stefansson et al., 2008; Weiss et al., 2008; Ben-Shachar et al., 2009; Doornbos et al., 2009; McCarthy et al., 2009). By contrast, autism-associated CNVs, such as deletions at 16p11.2 (Kumar et al., 2008), or duplications at 22q11.21 (Glessner et al., 2009; Crespi et al., 2009) have seldom also been reported in individuals diagnosed with schizophrenia, which suggests that false-positive diagnoses of schizophrenia as autism are uncommon.

Differentiating between a hypothesis of false-positive diagnoses of premorbidity to schizophrenia as autism, compared to a hypothesis of specific deletions or duplications shared between autism and schizophrenia, requires some combination of longitudinal studies, judicious use of endophenotypes, and adoption of relatively new diagnostic categories such as multiple complex developmental disorder (Sprong et al., 2008). Moreover, to the degree that such false positives are not uncommon, and autism and schizophrenia are underlain by diametric genetically based risk factors, inclusion of children premorbid for schizophrenia in studies on the genetic bases of autism will substantially dilute the probability of detecting significant results.

Ultimately, robust evaluation of alternative hypotheses for the relationship of autism with schizophrenia will require evidence from studies of common and rare SNP variants as well as CNVs, in-depth analyses of the neurodevelopmental and neuronal-function effects of different alterations to genes such as NRXN1, CNTNAP2, and SHANK3, and integrative data from diverse disciplines other than genetics, especially the neurosciences and psychology. Unless such interdisciplinary studies are deployed—in hypothesis-testing frameworks that use strong inference—we should expect to remain, as penned by García Márquez, in “permanent alternation between excitement and disappointment, doubt and revelation, to such an extreme that no one knows for certain where the limits of reality lay”—for yet another 100 years.

The Diametric Opposition of Autism and Psychosis: Support From a Study of Cognition
As has been noted previously, Crespi and Badcock’s (2008) theory that autism and schizophrenia are diametrically opposed disorders is certainly a novel and somewhat controversial one. In his recent blog on Psychology Today, Badcock states that the theory stands on two completely different foundations: one in evolution and genetics, and one in psychiatry and cognitive science (Badcock, 2010). While most of the comments posted before ours have addressed the relationship between autism and schizophrenia from a genetic perspective, coming from a psychology background, we note that it is the aspects of Crespi and Badcock’s theory that relate to cognition which have particularly caught our attention. While we can therefore contribute little to the discussion of a relationship between autism and schizophrenia from a genetic standpoint, we present the findings from our recent study (Russell-Smith et al., 2010), which provided the first test of Crespi and Badcock’s claim that autism and psychosis are at opposite ends of the cognitive spectrum.

In placing autism and psychosis at opposite ends of the cognitive spectrum, Crespi and Badcock (2008) propose that autistic and positive schizophrenia traits contrastingly affect preference for local versus global processing, with individuals with autism displaying a preference for local processing and individuals with positive schizophrenia displaying a preference for global processing. That is, these authors claim that while individuals with autism show a tendency to focus on detail or process features in their isolation, individuals with positive schizophrenia show a tendency to look at the bigger picture or process features as an integrated whole. Importantly, since Crespi and Badcock argue for a continuum stretching all the way from autism to psychosis, the same diametric pattern of cognition should be seen in individuals who display only mild variants of autistic and positive schizophrenia traits. This includes typical individuals who score highly on measures such as the Autism Spectrum Quotient (AQ; Baron-Cohen et al., 2001) and the Unusual Experiences subscale of the Oxford-Liverpool Inventory of Experiences (O-LIFE:UE; Mason et al., 2005). These are both reliable and commonly used measures which have been specifically designed to assess the levels of “autistic-like” traits and positive schizotypy traits in typical individuals. Since Crespi and Badcock actually argue their theory is best evaluated with reference to individuals with milder traits of autism and positive schizophrenia, it is with these populations that we investigated their claims.

A task often used to determine whether an individual has a preference for local over global processing is the Embedded Figures Test (EFT; Witkin et al., 1971), which requires individuals to detect hidden shapes within complex figures. As the test requires one to resist experiencing an integrated visual stimulus or gestalt in favor of seeing single elements, it is argued that a local processing style aids successful (i.e., faster) completion of this task (Bolte et al., 2007). Accordingly, from Crespi and Badcock’s (2008) theory, one would expect that relative to individuals with low levels of these traits, individuals with high levels of autistic-like traits should perform better on the EFT, while individuals with positive schizotypy traits should perform worse. To test this claim, our study obtained the AQ and O-LIFE:UE scores for 318 students completing psychology as part of a broader degree (e.g., a BSc or BA). Two pairs of groups (i.e., four groups in total), each consisting of 20 students, were then formed. One of these pairs consisted of High and Low AQ groups, which were selected such that they were separated substantially in their AQ scores but matched as closely as possible on their O-LIFE:UE scores. The other pair of groups, the High and Low O-LIFE:UE groups, were selected such that they were separated in their O-LIFE:UE scores, but matched as closely as possible on their AQ scores. The gender ratio was matched closely across the four groups.

To test the prediction that higher levels of autistic-like traits are associated with more skilled EFT performance, the High and Low AQ groups were compared in terms of their mean response time to accurately locate each of the embedded figures. Individuals in the High AQ group did display more skilled EFT performance than individuals in the Low AQ group, consistent with a greater preference for local over global processing in relation to higher levels of autistic-like traits (see also Almeida et al., 2010; Bolte and Poustka, 2007; Grinter et al., 2009; Grinter et al., 2009). We then compared EFT performance for the O-LIFE:UE groups to test the prediction that higher levels of positive schizotypy traits are associated with less skilled performance on this task. Consistent with a preference for global over local processing in relation to higher levels of positive schizotypy traits, individuals in the High O-LIFE:UE group displayed less skilled EFT performance than individuals in the Low O-LIFE:UE group. Therefore, results from both pairs of groups together provide support for Crespi and Badcock’s (2008) claim that autistic and positive schizophrenia traits are diametrically opposed with regard to their effect on local versus global processing.

However, the support our study offers for Crespi and Badcock’s (2008) theory was tempered slightly by our failure to find the expected contrasting patterns of non-verbal relative to verbal ability for our two pairs of groups. To display the expected patterns, relative to individuals with low levels of these traits, individuals with high levels of autistic-like traits should have displayed higher non-verbal ability relative to verbal ability, whereas individuals with high levels of positive schizotypy traits should have displayed lower non-verbal relative to verbal ability. While visual inspection of mean verbal and non-verbal scores for the O-LIFE:UE groups revealed a trend consistent with what would be expected based on Crespi and Badcock’s theory, none of the group differences was statistically significant. However, as we pointed out in our article, a study which offers a more complete assessment of this aspect of the theory is warranted. In particular, since the use of a student sample in our study no doubt led to a restriction in the range of IQ scores (especially with reference to verbal IQ), a test of community-based samples would be useful.

Therefore, while Crespi and Badcock’s (2008) theory has passed its first major test at the level of cognition, with our results indicating a contrasting effect of autistic-like and positive schizotypy traits with regard to preference for local versus global processing, further investigation of these authors’ theory at both the cognitive and genetic levels is required.

Bolte, S., Poustka, F. (2006). The broader cognitive phenotype of autism in parents: How specific is the tendency for local processing and executive function. Journal of Child Psychology and Psychiatry, 47, 639-645. Abstract

De novo CNVs are associated with advanced paternal age in a mouse model
While the association between advanced paternal age and an increased risk of various neuropsychiatric disorders such as schizophrenia and autism is now well established, the mechanism underpinning this finding remains unclear. Putative mechanisms include de-novo mutations and/or epigenetic mechanisms. In light of the growing body of evidence linking copy number variants (CNVs) with these same disorders, we used a mouse model to explore the hypothesis that the offspring of older males have an increased risk of de-novo CNVs. C57BL/6J sires that were three- and 12-16 months old were mated with three-month-old dams to create control offspring and offspring of old sires, respectively. Applying genomewide microarray screening technology, seven distinct CNVs were identified in a set of 12 offspring and their parents.

Competitive quantitative PCR confirmed these CNVs in the original set and also established their frequency in an independent set of 77 offspring and their parents. On the basis of the combined samples, six de-novo CNVs were detected in the offspring of older sires, whereas none were detected in the control group. Two of the CNVs were associated with behavioral and/or neuroanatomical phenotypic features. One of the de-novo CNVs involved Auts2 (autism susceptibility candidate 2), and other CNVs included genes linked to schizophrenia, autism, and brain development.

Our results support the hypothesis that the offspring of older fathers have an increased risk of neurodevelopmental disorders such as schizophrenia and autism by generation of de-novo CNVs in the male germline.